Antitubercular drug resistance in four healthcare
facilities in North India.

Abstract:

Tuberculosis (TB) is a major public-health problem in India, having
the highest number of incident and multidrug-resistant (MDR) TB cases.
The study was carried out to appraise the prevalence of first-line
anti-TB drug resistance in Mycobacterium tuberculosis (MTB) and its
patterns among different types of TB patients from different settings in
a province of North India. Of 3,704 clinical specimens, 345 (9.3%) were
culture-positive, and drug-susceptibility testing was carried out for
301 MTB strains. A high level of primary and acquired drug resistance of
MTB was observed in the region studied, with weighted mean of 10.5% and
28.08%, 12.81% and 29.72%, 17.12% and 29.94%, 11.97% and 27.84%, and
10.74% and 23.54% for rifampicin, isoniazid, streptomycin,
ethambutol-resistant and MDR cases respectively. Drug resistance was
significantly higher in pulmonary (p=0.014) and acquired drug-resistant
TB cases (p<0.001). Any drug resistance (p=0.002) and MDR TB were
significantly (p=0.009) associated with HIV-seropositive cases. An
urgent plan is needed to continuously monitor the transmission trends of
drug-resistant strains, especially MDR-TB strains, in the region.

India has the highest number of the incident tuberculosis (TB) and
multidrug-resistant (MDR) TB cases; yet the factors contributing to
emergence, spread, and containment of TB are not well-estimated. TB is a
major public-health problem, particularly in developing nations where
the prevalence of infection is 40% (1). The incidence has been
accelerated by the HIV epidemic, the appearance of new genotypes,
multidrug-resistant and extensively drug-resistant (XDR) strains of
Mycobacterium tuberculosis (MTB). Development of drug resistance in the
population has increased the possibility that TB may once again become
an incurable disease.

In 2008, there were 9.4 million new TB cases (including 3.6 million
women and 1.4 million cases among people living with HIV) throughout the
world. Approximately 1.8 million people died from TB in 2008, of whom
500,000 were HIV-infected people. HIV-positive people co-infected with
TB are 20-40 times more likely to develop active TB than people without
HIV infection living in the same country (2).

In 2008, most of the estimated number of TB cases occurred in Asia
(55%) and Africa (30%). The five countries ranking first to fifth in
terms of total numbers of incident cases were India (1.6-2.4 million),
China (1-1.6 million), South Africa (0.380.57 million), Nigeria
(0.37-0.55 million), and Indonesia (0.34-0.52 million). An estimated 35%
of TB cases worldwide were found in India and China alone. There were an
estimated 0.5 million cases of MDR-TB worldwide in 2007. The countries
that ranked first to fifth in terms of total numbers of MDR-TB cases in
2007 were India (n=1,31,000), China (n=1,12,000), the Russian Federation
(n= 43,000), South Africa (n=16,000), and Bangladesh (n=15,000). To meet
the targets set in the global plan, diagnosis and treatment of MDR-TB
need to be rapidly scaled up, especially in the three countries that
account for 57% of global cases: China, India, and the Russian
Federation (3).

The incidence of TB is the greatest among those with impaired
immunity, such as people with HIV infection and diabetes. HIV is a very
important risk factor that enhances the progression of active TB in
people with latent TB infection (4). The lifetime risk of TB in
immunocompetent persons is 5-10% but, in HIV-positive individuals, there
is a 5-15% annual risk of developing active TB disease (5). Diabetes
mellitus (DM), a metabolic disorder, weakens the immune system. The
incidence of TB, particularly pulmonary TB (PTB), is higher in diabetics
compared to non-diabetics (6).

Resistance in cultures from patients for which treatment had been
administered for <1 month or not at all is known as primary drug
resistance (PDR) while that from patients with one or more previous TB
treatment episodes (for at least one or more than one month), including
those with treatment failures and relapse, is called acquired drug
resistance (ADR) (7). MDR-TB is defined as resistance to the two main
first-line anti-TB drugs--isoniazid (INH) and rifampicin (RIF)--with or
without resistance to any other drugs. XDR-TB is a form of TB caused by
bacteria that are resistant to most effective anti-TB drugs and defined
as resistance to at least RIF and INH and to any member of the quinolone
family and at least one of the following second-line anti-TB injectable
aminoglycosides: kanamycin, capreomycin, or amikacin (8). Both primary
drug resistance and acquired drug resistance contribute to MDR/XDR TB
(9).

MDR-TB is an emerging problem in the world. Several outbreaks of
MDR-TB have recently been reported. The prevalence of MDR-TB in India is
3.4% in primary (new) TB cases and 25% in acquired cases (10). In the
second global report of the World Health Organization/International
Union Against Tuberculosis and Lung Disease followed in 2000 in 58
countries, the median prevalence of resistance to at least one drug
among new TB cases was 10.7% (range 2-36%), and that of MDR-TB was 1%
(range 0-14%). In the previously-treated cases, the median prevalence of
resistance to at least one drug was 23% (range 0-94%) and that of MDR-TB
was 9% (range 0-48%) (11,12).

Surveillance data on primary and acquired drug resistance in MTB
are important to design TB-control programmes. Escalating HIV infection
and diabetes and negligence in TB control have caused an increase in the
incidence of TB over the last decade in both developing and developed
countries (13,14). Moreover, several other factors, such as
homelessness, poverty, lack of infrastructure in public health, and
inadequate access to health services have played an important role in
worsening the situation.

In the present study, we aimed at determining the prevalence of
first-line anti-TB drug resistance and its patterns in MTB isolated from
different types of TB patients of North India.

MATERIALS AND METHODS

Study settings

The study was conducted at the Department of Microbiology,
Institute of Medical Sciences, Banaras Hindu University (BHU). Sir
Sundar Lal Hospital, a tertiary-care hospital of BHU, has a vast
catchment area, this being the only tertiary-care hospital in
north-eastern Uttar Pradesh (UP) providing medical coverage to a
population of over 15 crore in eastern UP, western Bihar, and adjoining
areas of Madhya Pradesh and Nepal. Our mycobacteriology laboratory is
equipped to perform culture and drug-sensitivity testing (DST) for MTB.
The sputum samples were collected from selected TB centres based on the
maximum frequency of patients attending those centres. These were
Department of TB and Respiratory Disease and antiretroviral therapy
(ART) centres of Sir Sundar Lal Hospital of BHU, Shree Shiv Prasad Gupta
District Hospital, Kabir Chaura (a secondary-care centre), Swami
Vivekanand Smarak Rajkiya Chikitsalaya, Bhelupura (a primary healthcare
unit), and Integrated Counselling and Testing Centre (ICTC) of the
Department of Microbiology, Institute of Medical Sciences, BHU (a
tertiary-care centre). The duration of the study was 25 months from
January 2008 to January 2010. It included samples from both inpatients
and outpatients.

Study subjects

The study included TB patients with or without any other additional
complication, such as HIV-seropositivity/diabetes (based on their
previous and current medical records). Information was collected from
the medical files and compliance charts on demographic characteristics
of patients, radiological studies, and sputum mycobacteriologic studies.

Collection and transportation of specimens

Specimens were collected in disposable wide-mouthed containers
which were made of clear thin plastic, unbreakable and leak-proof
material. These were placed in a box which could withstand leakage of
contents, shocks, and other conditions incident to ordinary handling
practices. Those boxes were immediately transported to the laboratory.

Specimens/mycobacterial strains

Both pulmonary and extra-pulmonary specimens from 3,704
clinically-suspected TB patients, i.e. sputum, bronchoalveolar lavage
(BAL), gastric fluid and cerebrospinal fluid (CSF), endometrial tissues,
pus, fine-needle aspirate, urine, pleural fluid, lymphnode biopsy,
ascitic fluid, pericardial fluid, knee fluid, and sinus discharge, were
collected and used for preparing smear. The smears were subjected to
acid fast stain by Ziehl-Neelsen method and examined by light microscopy
at 100x oil-immersion objective. Sputum, BAL, pus and urine specimens
were decontaminated by modified Petroff's method using 4% NaOH
(15,16) while decontamination of other specimens was not needed as those
were collected aseptically. All the specimens were concentrated by
centrifugation at 3,200 x g for 20 minutes. The supernatant was
discarded, and a part of the sediment was used for culture. Isolated
cultures were characterized by certain biochemical tests, such as
heat-stable catalase, niacin accumulation, and susceptibility to p-nitro
benzoic acid (PNB) (17).

Drug-sensitivity testing

Isolated MTB strains were subjected to indirect DST by proportion
method (PM), the gold standard for DST of MTB. Conventional
Lowenstein-Jensen (LJ) medium was prepared as described earlier (15).
DST was carried out on LJ medium according to the standard procedures of
the laboratory, with the recommended critical concentrations of 40
[micro]g/mL for RIF, 0.2 [micro]g/mL for INH, 2 [micro]g/mL for
ethambutol (EMB), and 4 [micro]g/mL for streptomycin (STR) (15,18). In
brief, bacterial suspension for DST was prepared in the concentration of
1 mg/mL suspension (S1 suspension). S1 was further diluted 10-fold to
obtain S2-S4. S1-S4 bacterial concentrations were respectively
inoculated into drug-free and drug-containing LJ slopes using a 3-mm
internal diameter wire-loop and incubated at 37[degrees]C. Growth was
recorded at 28 days and at 42 days as follows: +++ for confluent growth,
++ for more than 100 colonies, and 1-100 actual numbers of colonies.
Susceptibility or resistance was recorded when the proportion of
bacteria in drug-containing medium to that of drug-free medium was <1
or >1 respectively.

Laboratory quality control/quality assurance

H37Rv (ATCC 27294) and a known MDR strain were used as controls.
The laboratory supervisor examined the DST results. DST of 69 MTB
strains (23%), which showed contamination, was repeated.

Analysis of data

The proportion of resistant isolates per setting was calculated.
The weighted mean for each setting was calculated by multiplying the
number of resistant cases in each setting and the weighted case (number
of cases in each setting divided by the total number of cases) of the
same setting. Other results were analyzed with the SPSS software
(version 12.0.1) (SPSS Inc., Chicago, IL, USA). The features of two
groups were compared using the Z-test and of three groups by chi-square
([chi square]) test for the assessment of statistical significance. A p
value of <0.05 was considered significant.

Ethical issues

The study was approved by the ethical committee of the institution.

RESULTS

Pulmonary (85.56%) and extra-pulmonary (14.44%) specimens from
3,704 clinically-suspected TB patients were collected. Statistically, a
sample-size of approximately 3,704 was required for investigation of the
prevalence of TB and MDR-TB in our region.

Of the 3,704 specimens, 345 (9.3%) were culture-positive, of which
333 (96.52%) were tubercle, and 12 (3.48%) were non-tubercle bacilli. Of
the 333 MTB isolates, DST of only 301 was performed. Due to
contamination, we were unable to read the DST results of 32
culture-positive MTB isolates. Further, the sufficient extent of MTB
growth was not available to repeat DST.

Drug resistance

DST for all the four first-line anti-TB drugs, i.e. RIF, INH, STR,
and EMB, was performed. There were no significant differences between
the resistance rates of STR (46.84%), INH (42.83%), RIF (38.53%), EMB
(39.20%), and MDR (34.55%) (Fig.).

Primary, acquired and total drug resistance levels for RIF, INH,
STR, and EMB are shown in Table 1. Almost similar differences were found
in resistance proportions for the individual drug among all four
settings. On average, resistance proportions in new and acquired
resistant cases were the highest for STR (17.12% and 29.94%
respectively) and INH (12.81% and 29.72% respectively) whereas for EMB
and RIF, these were 11.97% and 10.55%, and 27.84% and 28.08%
respectively.

Some differences were observed in the percentage of MDR prevalence.
Among the new cases, it was the highest (25%) in the cases from the
first and third settings, followed by the fourth (15.0%) and second
(14.28%) settings. In the acquired cases, it was the highest in the
second setting (68.82%), followed by the fourth (66.66%) and third
(50.0%) settings whereas it was the lowest in the first one (45.45%)
(Table 1).

[FIGURE OMITTED]

Of the 301 cases studied, 199 (66.11%) were male, and 102 (33.88%)
were female. Of the 199 males, 104 (61.53%) showed resistance to one or
more than one tested anti-TB drugs whereas 65 (38.46%) of the 102
females showed drug-resistant TB. The mean age at diagnosis of
drug-resistant TB was 33 (range 4-75) years. Most cases of PTB were aged
21-40 years. Extra-pulmonary TB was documented mostly in the age-group
of <10 years. Most common extra-pulmonary TB found was tubercular
lymphadenitis (17.69%), followed by pleural TB (13.85%).

In the present study, resistance to any drug and multidrug
resistance were analyzed against sex, age, nature of specimen, and type
of resistance. Resistance was significantly higher in pulmonary
(p=0.014) and ADR-TB cases (p<0.001). However, resistance was not
significantly associated with any age-group or sex. In addition, MDR-TB
was significantly higher only in the case of ADR-TB (p=0.0019) (Table
2).

Of the 301 cases for which DST was performed, 269 (89.37%) were
both smear- and culture-positive whereas 32 (10.63%) cases were
smear-negative but culture-positive. Of the 269 cases, which were both
smear- and culture-positive, 151 (56.13%) were resistant to one or more
tested anti-TB drugs. Of the 32 culture-positive cases, 18 (10.65%) were
resistant to one or more tested anti-TB drugs. The prevalence of
resistance to any drug and multidrugs was analyzed for the same
categories and had no significant association (Table 3).

Of the 3,704 specimens, 82 (2.21%) were HIV-seropositive. Of the 82
HIV-seropositive specimens, 37 (45.12%) were culture-positive. Of the 37
culture-positive specimens, susceptibility testing was done for 32
specimens. By excluding the diabetic cases, the association of HIV and
TB was analyzed in 294 (97.67%) cases. Of the 294 cases, 32 (10.88%)
consecutive patients of TB were co-infected with HIV while 262 (89.12%)
were only culture-positive for TB. Of the 32 HIV-TB cases, 26 (15.85%)
were resistant to one or more tested anti-TB drugs, of which 10 (10.0%)
were MDR. The prevalence of resistance to any drug and multidrug
resistance was analyzed for the above two categories, and significant
associations were observed (Table 3).

Of the 3,704 specimens, 26 (0.70%) cases were diabetics, along with
smear-positivity. Of the 26 cases, only nine (34.62%) were
culture-positive, and susceptibility testing was done for eight strains.
By excluding the HIV cases, the association between diabetes and TB was
analyzed only in 270 (89.70%) cases. Of the 270 cases, eight (2.96%)
consecutive patients of TB had diabetes-related complication. Of the
eight diabetic-TB cases, six were resistant to one or more anti-TB
drugs, in which five were MDR. The prevalence of resistance to any drug
and multidrug resistance was analyzed for the above two categories, and
no significant association was found between them (Table 3).

DISCUSSION

DST is performed for several purposes, such as in relapse or
retreatment cases, to change the drug regimen when resistance is
suspected, or for undertaking drug resistance surveillance studies in a
region/country.

Considering the resistance in all the four settings, on average, it
was the highest for STR in new (17.12%) and in previously-treated cases
(29.94%) while it was the lowest for RIF (10.55%) in new cases and for
EMB (27.84%) in the previously-treated cases (Table 1). The same results
were observed in a study in Russia, in which the highest resistance was
observed for STR in new and previously-treated cases with 40.4% and
66.7% respectively whereas resistance to RIF and EMB was the lowest in
new cases (13.5%) and in the previously-treated cases (60.0%)
respectively (19). These observations clearly demonstrate the
significance of critical monitoring of drug resistance pattern in a
set-up, particularly where there is a high prevalence of drug
resistance.

According to the World Health Organization (WHO), India is number
one in terms of the prevalence of TB, and 3.4 million (17%) TB patients
have developed multidrug resistance. Drug resistance surveys in Gujarat
and Maharashtra (2005-2006) showed the prevalence of MDR-TB to be almost
3% among primary and 12-18% in previously-treated cases. It is estimated
that the prevalence of MDR-TB may be three times greater than its
incidence (20). MDR-TB in retreatment patients varies from 30% to 80% in
different regions (21). In this study, on average, the resistance
proportion of MDR-TB was high, with 34.61% among all the cases, 19.82%
showing primary resistance, and 57.73% acquired resistance. We report a
high rate of multidrug resistance in both newly-infected and
previously-treated cases, which is in good agreement with earlier
observations reported from India and some former Soviet Union countries
(22-24). Among the new cases, the percentage of MDR prevalence was the
highest (25%) in the cases from the first and third settings. In the
case of the first setting, it could be because all the extra-pulmonary
TB cases were from the same setting. In the third setting, being a
primary healthcare centre, the prevalence of MDR-TB was the highest in
new cases. In the acquired cases, it was the highest in the second
setting (68.82%), followed by the fourth setting (66.66%). The second
setting is a tertiary referral hospital, and the fourth setting is a
secondary healthcare centre (district TB centre, Varanasi, India) where
patients with more serious conditions may have presented, resulting in
the highest number of acquired resistant cases. This study noted that
the prevalence of MDR-TB was not significantly associated with age, sex,
pulmonary TB, or extra-pulmonary TB.

Furthermore, resistance to any drug was significantly higher in the
previously-treated patients (Table 2). The relationship between history
of receiving anti-TB treatment and drug resistance has been clearly
described in several studies (12,25,26). A significant difference in
drug resistance between the new and the retreatment patients confirms
the inefficiency of TB-control programmes. The use of irregular/improper
anti-TB drugs during recent years has led to accumulation and
multiplication of resistant strains. Notably, resistance to RIF, which
did not show an increase in the new cases, was significantly elevated in
the retreated cases. This reveals the fact that, with irregular
treatment and in the presence of INH resistance, virtual monotherapy
results in resistance to other agents as well (27).

The proportions of drug resistance among the new and the
previously-treated TB cases are important indicators for epidemiology of
TB. The level of initial drug resistance is said to be an
epidemiological marker to assess the success of the National TB
Programme. This also influences the design of regimens to be employed
and policy decisions. In a well-functioning TB-control programme, a low
level of mistake in treatment can increase the probability of high
resistance level among acquired resistant cases because drug resistance
is a strong risk factor for recurrent TB. However, if a good TB-control
programme is in place, proportion of the previously-treated patients
among all TB patients should be low.

Overall, we found that the drug resistance to STR and INH in the
new and the previously-treated cases was more frequent compared to other
agents. Similarly, the first, second and third rounds of the WHO Global
Projects and similar studies in Iran (eight years of surveillance) and
Thailand have shown that the resistance to the above-mentioned agents
was more common compared to the resistance to other first-line drugs
(1,12,25,28). In general, resistance to STR and INH has been reported to
be higher than EMB and RIF all over the world (29). However, other
patterns of anti-TB resistance also exist. In a 15-year surveillance in
Saudi Arabia, resistance to INH and EMB was more frequent than to other
first-line drugs (30) whereas, in a study in Dhaka, Bangladesh,
resistance to INH and RIF was found to be more frequent (31). Moreover,
a prospective study set in the National Masan Tuberculosis Hospital in
Masan, Republic of Korea, reported the enrollment and treatment of 19
patients with well-localized, cavitary pulmonary MDR-TB or XDR-TB with
anti-TB therapy consisting of INH, RIF, EMB, pyrazinamide (Z), and STR.
All recovered isolates of MTB were resistant to INH and RIF. Resistance
to the first-line agents--EMB, STR, and Z--was observed in 73.7%, 36.8%,
and 26.3% of isolates respectively (32).

In the present study, the prevalence of HIV in TB patients was
10.72% (37/345). A study in sub-Saharan Africa has recorded the
HIV-seroprevalence rates of 50-70% in patients with TB (33). However,
India has reported the HIV-seropositivity rates of 0.4-20.1% (34). This
highlights the importance of effective guidelines developed by the WHO
to control the emergence of TB co-infection in HIV/AIDS. In our study,
resistance to any drug was significantly higher in the HIV-infected
patients compared to the non-HIV patients. The same trend was also
observed in the case of MDR-TB. According to the literature, infections
with HIV and alcohol-abuse are important risk factors for the
development of TB and the appearance of drug resistance in MTB infection
(35).

The incidence of TB is higher in diabetics compared to the general
population. The risk of developing an active TB infection is 3-7 times
greater in persons with diabetes than non-diabetics (36). Each year,
India accounts for one-fifth of the newly-diagnosed TB cases worldwide,
of which almost half have diabetes (37). The prevalence of diabetes
worldwide is close to 10%, and the relative risk of TB varies from 3% to
>8% depending on the study (38). Furthermore, Stevenson et al.
concluded that, in India, diabetes makes a substantial contribution to
the burden of incident TB (39), and the association is particularly
strong for the infectious form of TB. However, in our study, there was
no significant relationship between TB and diabetes, which might be due
to the enrollment of a low number of cases. The overall importance of
diabetes as a risk factor for TB is still largely unidentified, although
a recent study in Mexico concluded that, in the population studied, 25%
of pulmonary TB was attributable to diabetes (36).

Our centre is an important referral centre in North India. However,
our data may not necessarily be representative of the national
population, especially with regard to new cases because only patients
with more serious conditions may have presented to our centre. However,
its findings will be indicative of the local population-related
information for the previously-treated patients. A nationwide survey of
drug resistance is required to achieve a more accurate assessment,
management, and control of this deadly infectious disease.

A strong and cost-effective TB-control programme can reduce the
incidence of drug resistance in the community. Some modifications should
be made in running of the TB-control programme. For ex ample, routine
quality-assured DST for those patients who are at a high risk of
resistance, especially failure cases, and those treated with second-line
drugs should be done. In addition, we felt that the screening of all
HIV/diabetic patients for TB and all TB patients for HIV and diabetes
will help detect co-infected patients who require treatment for both the
infections. This can be done by a good coordination and communication
between TB and AIDS/diabetes-control programmes. These have potentially
serious implications for TB control, and it must become a priority to
use the existing knowledge about the association of TB with HIV/
diabetes patients to initiate focused and coordinated actions, including
new research in parts of the world where diabetes/HIV is epidemic and TB
is endemic to properly inform public health and clinical practice.
Standard chemotherapy with individualized drug resistance therapy,
guided by conventional DST, might not be sufficient to control
drug-resistant TB in northern India. Therefore, there is an urgent
requirement of a plan to expand appropriate diagnostic and treatment
services for patients with drug-resistant TB, especially MDR-TB, with or
without having any additional complications, such as HIV/diabetes,
throughout India and the world.

ACKNOWLEDGEMENTS

The authors thank Dr. Susan van den Hof, KNCV Tuberculosis
Foundation, The Hague, The Netherlands and Dr. Guangxue HE, National
Center for TB Control and Prevention, China Center for Disease Control
and Prevention, Beijing, PR China, for their valuable guidance in
statistics.